The present invention relates to the use of 2-amino-4-pyridylmethyl thiazoline derivatives of formula (I): 
or pharmaceutically acceptable salts thereof as inhibitors of inducible NO-synthase.
The subject of the invention is the use of 2-amino-4-pyridylmethyl thiazoline derivatives of formula (I) and pharmaceutically acceptable salts thereof for the preparation of pharmaceutical compositions intended for preventing and treating diseases in which an abnormal production of nitric oxide (NO) by induction of inducible NO-synthase (NOS-2 or iNOS) is involved, the pharmaceutical compositions containing the novel 2-amino-4-pyridylmethyl-thiazoline derivatives and the pharmaceutically acceptable salts thereof and the novel 2-amino-4-pyridylmethyl-thiazoline derivatives and pharmaceutically acceptable salts thereof.
Nitric oxide (NO) is a diffusable radical involved in many physiological and pathological processes. It is synthesized by oxidation of L-Arginine, a reaction catalyzed by a family of enzymes known as nitric oxide synthases or NO-Synthases (NOSs), referenced in the international enzyme nomenclature under the number E.C. 1.14.13.39.
Three NOS isoforms, two of which are constitutive and one inducible, are known:
a neuronal NOS (NOS-1 or nNOS) was originally isolated and cloned from nerve tissue in which it is a constitutive enzyme. The NOS-1 produces NO in response to various physiological stimuli such as the activation of membrane receptors according to a mechanism dependent on calcium and on calmodulin.
an inducible NOS (NOS-2 or iNOS) can be induced in response to immunological stimuli such as, for example, cytokines or bacterial antigens in various cells such as, for example, macrophages, endothelial cells, hepatocytes, glial cells, as well as many other types of cells. This isoform activity is not regulated by calcium. Consequently, once induced, it produces a large amount of NO over prolonged periods.
an endothelial NOS (NOS-3 or eNOS) is constitutive and calcium/calmodulin dependent. It was originally identified in vascular endothelium cells, in which it generates NO in response to physiological stimuli such as the activation of membrane receptors.
The NO produced by the neuronal endothelial constitutive isoforms (NOS-1 and NOS-3) is generally involved in intercellular signalling functions. For example, the endothelial cells which line the inner wall of blood vessels induce the relaxation of the underlying smooth muscle cells via the production of NO. It thus contributes towards regulating the arterial pressure.
The NO produced in large amount by the inducible isoform NOS-2 is, inter alia, involved in the pathological phenomena associated with acute and chronic inflammatory processes in a large variety of tissues and organs.
An excessive production of NO by induction of NOS-2 thus plays a part in degenerative pathologies of the nervous system such as, for example, multiple sclerosis, focal or global cerebral ischemia, cerebral or spinal trauma, Parkinson""s disease, Huntington""s disease, Alzheimer""s disease, amyotrophic lateral sclerosis, migraine, depression, schizophrenia, anxiety, epilepsy. Similarly, aside the central nervous system, the induction of NOS-2 is involved in many pathologies with inflammatory components such as, for example, diabetes, atherosclerosis, myocarditis, arthritis, arthrosis, asthma, inflammatory bowel diseaese, Crohn""s disease, peritonitis, gastroesophageal reflux, uveitis, Guillain-Barrxc3xa9 syndrome, glomerulo-nephritis, lupus erythematosus and psoriasis. The NOS-2 was also involved in the growth of certain forms of tumors such as, for example, epitheliomas, adenocarcinomas or sarcomas, and in infections with Gram-positive or Gram-negative intracellular or extracellular bacteria.
In all the situations in which an overproduction of NO is deleterious, it thus appears to be desirable to reduce the production of NO by administering substances capable of inhibiting the NOS-2. However, given the important physiological roles played by the constitutive isoform NOS-3, in particular, in regulating the arterial pressure, it is essential that the inhibition of the isoform NOS-2 has the least possible effect on the isoform NOS-3. Actually, it is known that the administration of unselective inhibitors of NOS isoforms leads to vasoconstriction and an increase in arterial pressure (Moncada, S., Palmer, R. M. J. and Higgs, E. A., Biosynthesis of nitric oxide from L-arginine: a pathway for the regulation of cell function and communication, Biochem. Pharmacol., 1989, 38: 1709-1715). These effects on the cardiovascular system are deleterious since they reduce the supply of nutrients to the tissues. Consequently, the present invention relates to compounds whose inhibitory activity with respect to NOS-2 is significantly higher than their inhibitory activity with respect to NOS-3.
Thiazoline-based NOS inhibitors are described in particular in patent applications WO94/12165, WO95/11231 and WO96/14842.
Thus in accordance with the present invention there is provided a series of 2-amino-4-pyridylmethyl-thiazoline derivatives of formula (I): 
wherein either R1, R2 are identical and represent an hydroxy radical, (C1-C4)alkyl, chlorine or (C1-C4) alkoxy; or at least one of R1 or R2 is hydrogen and the other is a (C1-C4) alkyl radical, (C1-C4) alkoxy, hydroxy or chlorine. The present invention also provides the preparation of useful medicinal products comprising a compound of formula (I) for preventing or treating the diseases in which an abnormal production of nitric oxide (NO) by induction of inducible NO-synthase (NOS-2 or iNOS) is involved.
In the above definitions and in those which follow, the (C1-C4)alkyl radical and (C1-C4)alkoxy contain 1 to 4 carbon atoms in straight or branched chain.
The compounds of formula (I) contain one or more asymmetric carbons and can thus be in racemic form or in the form of enantiomers and diastereoisomers; these also form a part of the invention as well as the mixtures thereof.
Moreover, the compounds of formula (I) can be in the tautomeric form (Ia): 
These tautomers also form a part of the invention.
Among the compounds of formula (I) useful according to the invention, mention may be made of the following compounds:
4-(2-hydroxy-pyridin-4-ylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine,
4-(2-chloro-pyridin-4-ylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine, and
4-(2,6-dichloro-pyridin-4-ylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine.
Also included as part of this invention are the racemates, enantiomers, diastereoisomers thereof and the tautomers thereof, as well as pharmaceutically acceptable salts thereof, and more particularly the following compounds:
(+)-4-(2-hydroxy-pyridin-4-ylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine,
4-(2-chloro-pyridin-4-ylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine,
4-(2,6-dichloro-pyridin-4-ylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine, and
the tautomers thereof, as well as the pharmaceutically acceptable salts thereof.
Among the compounds of formula (I) useful according to the invention and particularly preferred, mention may be made of the following compound:
4-(2-hydroxy-pyridin-4-ylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine, and
the racemates, enantiomers thereof and tautomers thereof, as well as the pharmaceutically acceptable salts thereof, and most particularly the following compound:
(+)-4-(2-hydroxy-pyridin-4-ylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine, and
the tautomer thereof as well as the pharmaceutically acceptable salts thereof.
The invention also relates to the pharmaceutical compositions containing, as active principle, a derivative of formula (I) for which either R1, R2 are identical and represent an hydroxy radical, a (C1-C4) alkyl, a chlorine, or a (C1-C4) alkoxy; or at least one of R1 or R2 is an hydrogen and the other is a (C1-C4) alkyl radical, (C1-C4) alkoxy, hydroxy or chlorine as well as the racemates, enantiomers, diastereoisomers thereof and mixtures thereof, tautomers thereof and pharmaceutically acceptable salts thereof.
The compounds of formula (I) can be prepared by cyclization of a derivative of formula: 
in which R1 and R2 have the same meanings as in formula (I) as defined above.
This cyclization is generally carried out using an acid such as hydrochloric acid, in an aqueous medium, at a temperature of about 100xc2x0 C. 6N hydrochloric acid is preferably used.
The derivative of formula (II) can be obtained according to the following reaction schemes: 
In these formulae Ra is a protecting group of the amine function such as those described by T. W. GREENE, Protective groups in Organic Synthesis, J. Wiley-Interscience Publication (1991), preferably an acetyl or tert-butoxycarbonyl radical, Rb is a (C1-C4) alkyl or alkoxycarbonyl radical, preferably, methyl, ethyl or isobutyloxycarbonyl and Hal is an halogen atom, preferably, chlorine, bromine or iodine.
The reaction a is generally carried out in the presence of a sodium (C1-C4) alkoxide (preferably sodium ethoxide), in the corresponding alcohol, at a temperature of between 10xc2x0 C. and the the boiling point of the reaction medium.
The reaction b is generally carried out in an inert solvent such as dimethylformamide in the presence of lithium iodide, at a temperature of between 100xc2x0 C. and the boiling point of the reaction medium or in a (C1-C4) aliphatic alcohol in the presence of sodium hydroxide at a temperature of between 10xc2x0 C. and 30xc2x0 C., followed by neutralizing with aqueous HCl (preferably 6N to 12N) then heating in a solvent such as dioxane or (C1-C4) aliphatic alcohol at a temperature of the boiling point of the reaction medium.
The reaction bxe2x80x2 is preferably carried out using 12 N hydrochloric acid at a temperature of about 100xc2x0 C.
The reaction bxe2x80x3 for the derivatives for which Rb is an alkyl radical is generally carried out by the action of a (C1-C4) aliphatic alcohol (preferably methanol or ethanol), in the presence of an inorganic acid such as sulfuric acid at a temperature of between 50xc2x0 C. and the the boiling point of the reaction medium. For the derivatives for which Rb is an isobutyloxycarbonyl radical, this reaction is generally carried out by the action of isobutyl chloroformate in the presence of a base such as triethylamine, in an inert solvent such as tetrahydrofuran at a temperature of between xe2x88x9220xc2x0 C. and 0xc2x0 C.
The reduction reaction c is preferably carried out using an hydride such as sodium borohydride or lithium aluminum hydride in a (C1-C4) aliphatic alcohol or tetrahydrofuran, at a temperature of between 10xc2x0 C. and 30xc2x0 C.
The deprotection reaction d for the compounds for which Ra is a protecting group of the amine function is carried out by any method of deprotection known by those skilled in the art and particularly those described by T. W. GREENE, Protective groups in Organic Synthesis, J. Wiley-Interscience Publication (1991). Preferably, when the protecting group is an acetyl radical, this reaction is carried out using aqueous hydrochloric acid at a temperature of about 100xc2x0 C. When the the protecting group is a tert-butoxycarbonyl radical, this reaction is carried out using hydrochloric acid in dioxane, at a temperature of about 20xc2x0 C.
The reaction e is carried out by the action of tert-butyl isothiocyanate, in an inert solvent such as (C1-C4) aliphatic alcohol (preferably methanol or ethanol), optionally in the presence of a tertiary amine such as triethylamine, at a temperature of between 20xc2x0 C. and the boiling point of the reaction medium.
The compound of formula (II) for which R1 and R2 are OH can be prepared by the same action from the compounds of formula (III) for which Z is a silyl radical, preferably tert-butyldimethylsilyl, the preparation is described in Synthesis 1994, 486 and Tetrahedron 1986, 42, 2725. 
The compoud of formula (I) for which R1 is OH and R2 is hydrogen may be prepared according to the following reaction scheme: 
In these formulae Ra is a protecting group of the amine function such as those described by T. W. GREENE, Protective groups in Organic Synthesis, J. Wiley-Interscience Publication (1991), preferably an acetyl or tert-butoxycarbonyl radical and Rb is a (C1-C4) alkyl or alkoxycarbonyl radical, preferably, methyl, ethyl or isobutyloxycarbonyl. Rc is a protecting group of the alcohol function such as those described by T. W. GREENE, Protective groups in Organic Synthesis, J. Wiley-Interscience Publication (1991), preferably a silyl and most particularly tert-butyldimethylsilyl radical.
The reaction a is generally carried out in the presence of a sodium (C1-C4) alkoxide (preferably sodium ethoxide), in the corresponding alcohol at a temperature of between 10xc2x0 C. and the boiling point of the reaction medium.
The reaction b is generally carried out in an inert solvent such as the dimethylformamide in the presence of lithium iodide, at a temperature of between 100xc2x0 C. and the boiling point of the reaction medium or in (C1-C4) aliphatic alcohol, in the presence of sodium hydroxide, at a temperature of between 10 and 30xc2x0 C. followed by neutralizing with an aqueous 6N HCl then heating in an inert solvent such as dioxane or alcohol such as ethanol at the boiling point temperature of the reaction medium.
The reaction bxe2x80x2 is preferably carried out using 12 N hydrochloric acid, at a temperature of about 100xc2x0 C.
The reaction bxe2x80x3 for the derivatives for which Rb is an alkyl radical is generally carried out by the action of a (C1-C4) aliphatic alcohol (preferably methanol, ethanol), in the presence of an inorganic acid such as sulfuric acid at a temperature of between 50xc2x0 C. and the the boiling point of the reaction medium. For the derivatives for which Rb is an isobutyloxycarbonyl radical, this reaction is generally carried out by the action of isobutyl chloroformate in the presence of a base such as triethylamine, in an inert solvent such as tetrahydrofuran at a temperature of between xe2x88x9220xc2x0 C. and 0xc2x0 C.
The reduction reaction c is preferably carried out using a hydride such as sodium borohydride or lithium borohydride in a (C1-C4) aliphatic alcohol and/or tetrahydrofuran, at a temperature range of from about 10xc2x0 C. to about 30xc2x0 C.
The protection reaction d is carried out by any of the hitherto known method of protection of the alcohol function such as those described by T. W. GREENE, Protective groups in Organic Synthesis, J. Wiley-Interscience Publication (1991), preferably using tert-butyldimethylsilyl chloride in the presence of a base such as tertiary amine (preferably diisopropylethylamine), in a solvent such as dichloromethane, at a temperature range of from about 0xc2x0 C. to about 30xc2x0 C.
The reaction e is carried out in the presence of 3-chloro-peroxybenzoxc3xafc acid, in a solvent such as dichloromethane at a temperature of between 0xc2x0 C. and the boiling point of the reaction medium.
The reaction f is carried out in the presence of para-toluenesulfonyl chloride in the presence of a base such as tertiary amine (preferably triethylamine), in a solvent such as alcohol (preferably methanol) at a temperature of between 10xc2x0 C. and the boiling point of the reaction medium.
The deprotection reaction g for the compounds for which Ra is a protecting group of the amine function and Rc is a protecting group of the alcohol function is carried out by any method of deprotection known by those skilled in the art and particularly those described by T. W. GREENE, Protective groups in Organic Synthesis, J. Wiley-Interscience Publication (1991). Preferably, when the protecting group Ra is an acetyl radical and when the the protecting group Rc is silyl and most particularly tert-butyldimethylsilyl radical, this is carried out using aqueous hydrochloric acid (preferably HCl 6N) at the boiling point temperature of the reaction medium.
The reaction h is carried out by the action of tert-butyl isothiocyanate, in an inert solvent such as (C1-C4) aliphatic alcohol (preferably methanol, ethanol), optionally in the presence of a tertiary amine such as triethylamine, at a temperature of between 20xc2x0 C. and the boiling point of the reaction medium.
The reaction i is generally carried out using an acid such as hydrochloric acid, in aqueous medium, at the boiling point temperature of the reaction medium. 6N hydrochloric acid is preferably used.
The compounds of formula (I) for which either R1 is OAlk and R2 is hydrogen, or R1 and R2 are OAlk can be respectively prepared from compounds of formula (I) for which either R1 is OH and R2 is hydrogen, or R1 and R2 are OH, by alkylation with a compound of structure Halxe2x80x94Alk. Hal represents an halogen atom (preferably chlorine, bromine or iodine) and Alk has the same meaning as in formula (I). This reaction is generally carried out in an inert solvent such as dimethylformamide, dimethylsulfoxide, dioxane, tetrahydrofuran in the presence of an acid acceptor such as trialkylamine (triethylamine for example), alkali metal hydroxide (sodium hydroxide, potassium hydroxide for example) or an alkali metal hydride (sodium hydride for example), at a temperature of between 20xc2x0 C. and the boiling point of the reaction medium.
The compounds of formula (I) are isolated and can be purified by the commonly known methods, for example by crystallization, chromatography or extraction.
The enantiomers of the compounds of formula (I) can be obtained by resolving the racemic mixtures, for example by chromatography on a chiral column according to PIRCKLE W. H. et al., Asymmetric Synthesis, vol. 1, Academic Press (1983) or by formation of salts or by synthesis from chiral precursors. The diastereoisomers can be prepared according to the known conventional methods (crystallization, chromatography or from chiral precursors).
The compounds of formula (I) may optionally be converted into addition salts with an inorganic or organic acid by the action of such an acid in an organic solvent such as an alcohol, a ketone, an ether or a chlorinated solvent. These salts also form a part of the invention.
Examples of pharmaceutically acceptable salts which may be mentioned are the following salts: benzenesulfonate, hydrobromide, hydrochloride, citrate, ethanesulfonate, fumarate, gluconate, iodate, isethionate, maleate, methanesulfonate, methylenebis-xcex2-oxynaphtoate, nitrate, oxalate, pamoate, phosphate, salicylate, succinate, sulfate, tartrate, theophyllinacetate and p-toluenesulfonate.
The compounds of formula (I) are inhibitors of NO-synthase inducible or NO-synthase of type 2 (NOS-2) and are thus useful for preventing and treating disorders associated with an excessive NO production such as mutiple sclerosis, focal or global cerebral ischemia, cerebral or spinal trauma, Parkinson""s disease, Huntington""s disease, Alzheimer""s disease, amiotrophic lateral scherosis, migraine, depression, schizophrenia, anxiety, epilepsy, diabetes, atherosclerosis, myocarditis, arthritis, arthrosis, asthma, inflammatory bowel disease, Crohn""s disease, peritonitis, gastro-esophageal reflux, uveitis, Guillain-Barrxc3xa9 syndrome, glomerulo-nephritis, lupus erythematosus and psoriasis, the growth of certain forms of tumors such as for example epitheliomas, adenocarcinomas or sarcomas, and infections with Gram-positive or Gram-negative intracellular or extracellular bacteria.
Their activities as inhibitors of NOS-2 and NOS-3 were determined by measuring the conversion of [3H]-L-arginine into [3H]-L-citrulline with, respectively, a NOS-2 enzymatic fraction extracted from the lungs of rats or mice pretreated with lipopolysaccharides (10 mg/kg i.p. 6 hours before collecting the tissue) and with a commercial preparation of recombinant bovine NOS-3. The compounds were incubated for 20 to 30 minutes at 370xc2x0 C. in the presence of 5 xcexcM (for NOS-2 activity) or 10 xcexcM (for NOS-3 activity) of [3H]-L-arginine, 1 mM of NADPH, 15 xcexcM of tetrabiopterine, 1 xcexcM of FAD, 0.1 mM of DTT in a HEPES buffer (50 mM, pH 6.7) containing 10 xcexcg/ml of calmoduline and 1.25 mM of CaCl2 when the NOS-3 activity was measured. The incubation was stopped by adding cold HEPES buffer (100 mM, pH 5.5) containing 10 mM EGTA and 500 mg of cationic ion-exchange resin (AG50W-X8, counter-ion: Na+) to separate the [3H]-L-arginine from the [3H]-L-citrulline. After separation of the phases by settling for 5 min, the radioactivity remaining in the liquid phase was measured in a scintillation counter in the presence of a suitable scintillating liquid. The yield for the recovery of the formed L-[3H]citrulline was able to be estimated using L-[ureido-14C]-citrulline as external standard.
The activity NOS-2 or NOS-3 was expressed in picomole(s) of [3H]-L-citrulline formed per minute and per milligram of protein contained in the reaction medium.
In this test on the enzyme NOS-2, the IC50 value for the compounds of formula (I) is less than or equal to 10 xcexcM.
The selectivity is measured by the IC50 NOS-3/IC50 NOS-2 ratio. This selectivity is greater than 45.
The compounds of formula (I) are of low toxicity. Their LD50 is greater than 40 mg/kg via cutaneous route in mice.